Using Scam to Investigate Reconfiguration of Molecular Determinants in D1-S6 during Slow Inactivation of hNav1.4

Abstract

The goals of this research are to determine if 1) a single point mutation affects gating kinetics and 2) whether MTS accessibility changes via inactivation. This will be accomplished using the substituted cysteine accessibility method (SCAM) by substituting cysteine for each of the target amino acids and by applying methanethiosulfonate ethylammonium (MTSEA), which selectively binds to cysteine, indicating when the sulfhydryl side chain is accessible to binding. The target mutants consist of positions from the gating hinge to near the bundle crossing in domain 1 - segment 6 (D1-S6). At this time, data for all mutants have been recorded except for Y437C & L438C. Patch clamp techniques were used to record whole-cell currents from transiently transfected HEK cells to determine 1) the potential effects of the cysteine substitution on activation, fast inactivation, and slow inactivation, and 2) the potential accessibility of cysteine in the closed, fast inactivated, and slow inactivated states. Several mutations affected one or more baseline kinetics of the sodium channel, including significant differences in the voltage needed for either fast inactivation and/or slow inactivation in N440C, L441C, G434C, and I439C. However, MTS exposure in mutants L433C, G434C, S435C, F436C, I439C, N440C, L441C, I442C, and L443C were found to have had little-to-no effect on when the mutants were in either the closed state (−160 mV), the fast-inactivated state (brief depolarization at 0 mV), or the slow-inactivated state (prolonged depolarization at 0 mV). Thus, our results suggest that changes in accessibility of the substituted cysteines in D1-S6 of hNav1.4 may not occur, and therefore that D1-S6 may not undergo prominent reconfiguration during slow inactivation gating.